In surface enhanced Raman scattering (SERS), substances in close proximity to nanometer-scale roughened noble metal surfaces exhibit large (up to 10.sup.6 -fold) enhancements in vibrational spectral intensities. When a laser used to excite SERS is in resonance with an electronic transition of the substance (surface enhanced resonance Raman scattering or "resonant SERS"), an additional 10.sup.3 -fold enhancement is seen. Accordingly, SERS has been utilized in a wide variety of applications, including detection of molecules, elaboration of structure and function of large biomolecules, and elucidation of chemistry occurring at metal, metal oxide, and polymer surfaces, to name a few.
The literature teaches many approaches for creation, fabrication, or assembly of structures with the requisite nanoscale roughness. These include evaporated metal films, aggregated colloidal metal sols, electrochemically-roughened macroscopic electrodes, and a host of others. In all these approaches, the substance being studied by SERS is placed in direct or close contact with the surface.
These substrates have been of limited utility for studies of biomolecules. For uncoated SERS substrates, there is typically biomolecule denaturation (Smulevich, G.; Spiro, T. G. J. Phys. Chem. 1985, 89, 5168-5173; Holt, R. E.; Cotton, T. M. J. Am. Chem. Soc., 1987, 109, 1841-1845). In constrast, biomolecules conjugated to colloidal Au nanoparticles retain their biological activity (Hayat M. A., Ed.; Colloidal Gold: Principles, Methods, and Applications, 3 vols.; Academic Press: New York, 1989), but the SERS intensities for such species by themselves have been weak (Garrell, R. L. Anal. Chem. 1989, 61, 410A-411A; Brandt, E. S.; Cotton, T. M. In Investigations of Surfaces and Interfaces-Part B; Rossiter B. W. and Baetzold R. C., Eds.; 2nd ed., John Wiley & Sons: New York, 1993; Chapter 8). Moreover, SERS has been of little value for studies of proteins whose chromophores are "buried" within the structure of the protein, and thus .quadrature.10 .ANG. away from the metal substrate. For example, cytochrome c.sub.3, a protein composed of four subunits that each contain a c-type heme moiety, exhibits SERS scattering for only one of the heme groups--that which is closest to the Ag substrate surface (Eng, L. H.; Schlegel, V.; Wang, D.; Neujahr, H. Y.; Stankovich, M. T.; Cotton, T. Langmuir 1996, 12, 3055-3059). This inability to observe SERS signals from even resonantly-enhanced chromophores within proteins results from the exponential dropoff in electromagnetic field away from the substrate surface. Finally, in the cases where SERS spectra can be obtained from adsorbed proteins, there is no control over protein orientation with respect to the surface, or over the conformational stability of the adsorbed protein over time.
An additional weakness of previously-described substrate architectures for SERS is their inability to rationally exploit what are known to be optimal SERS nanostructures. Early SERS work showed that electrochemical roughening of Ag electrodes in the presence of analyte molecules led to increased enhancements, a result verified by more recent experiments (Wolkow, R. A.; Moskovits, M. J. Chem. Phys. 1992, 96, 3966-3980; Gu, X. J.; Akers, K. L.; Moskovits, M. J. Phys. Chem. 1991, 95, 3696-3700.). This effect has been attributed to the entrapment of analyte molecules within the newly-formed Ag nanostructures. SERS studies using evaporated and cold-deposited Ag films suggested that the molecules responsible for the observed signal are located in crevices or pores in the films (Osawa, M.; Yamamoto, S.; Suetaka, W. Appl. Surf. Sci. 1988, 33/34, 890-897). In each system studied, it was postulated that the increased SERS signals resulted from the greatly enhanced electromagnetic (EM) fields that are possible between surface features. Theoretical calculations have shown that the electromagnetic fields in nanoparticle arrays are position-dependent. (Liver, N.; Nitzan, A.; Gersten, J. I. Chem. Phys. Lett. 1984, 111, 449-454).
There have been a number of studies in which the placement of the analyte relative to one SERS-active substrate has been controlled: using an organic or inorganic spacer, it is possible to control the distance between the analyte and SERS substrate over the zero .ANG.ngstrom (i.e. directly adsorbed) to few hundred .ANG.ngstrom regime (e.g. Ye, Q.; Fang, J.; Sun, L. J. Phys. Chem. B 1997, 101, 8221-8224). In this way, the relative contributions of the chemical enhancement and the electromagnetic enhancements to SERS have been elaborated, as well as the distance dependence of the latter.
Likewise, there have been a number of resonant SERS studies of the heme protein cytochrome c (Cc) directly adsorbed to SERS-active substrates (MacDonald, I. D. G.; Smith, W. E. Langmuir 1996, 12, 706-713; Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710-6721; Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6722-6728.; Sibbald, M. S.; Chumanov, G.; Cotton, T. M. J. Phys. Chem. 1996, 100, 4672-4678). This work has exploited the facts that the heme group of Cc is an intense chromophore and that the heme of Cc is very near the surface of the protein, at a cleft rich in positively-charged surface lysine residues. Since the surfaces of SERS-active surfaces are typically negatively-charged, this lysine-rich patch binds to the metal surface. To a first approximation, on vibrations perpendicular to the SERS substrate experience enhancement. However, in previous studies of Cc SERS, it has not been possible to control the angular orientation of the heme with respect to the SERS-substrate.
Ideally, an analyte would be sandwiched between two SERS-active substances in a known and controllable orientation. Moreover, it would be ideal if the relative contributions of the two substances could be elaborated, via the wavelength dependence of the SERS enhancements. Moreover, it would be ideal if such a sandwich could be demonstrated to yield improved stability toward deleterious changes in the structure of the analyte relative to the case where the analyte is directly adsorbed to one substrate. Furthermore, it would be ideal if such sandwiches could be prepared using vanishingly small quantities of the analyte; it would be of further benefit if it could be known that each molecule of analyte were attached to a least one member of the sandwich.
Accordingly, it is an object of this invention to provide a deliberately-prepared sandwich structure for SERS comprising an analyte complexed to a colloidal noble metal particle that is either adsorbed or covalently-attached to another SERS substrate.
It is another object of this invention to provide a deliberately-prepared sandwich structure for SERS in which the orientation of the analyte with respect to the two SERS-active components of the sandwich can be controlled.
It is a further object of this invention to provide a deliberately-prepared sandwich structure for SERS wherein the amount of analyte in which the amount analyte in the sample is exceedingly low.